Many complex behaviors, from language to navigation, consist of the sequential execution of smaller behavioral subunits. It remains poorly understood how animal brains orchestrate such sequences simultaneously at short and long timescales. Spider web-making offers a particularly interesting model in which to study complex sequential behaviors. Orb webs are built in five sequential and distinct phases of construction that collectively span multiple hours, requiring the coordination of subsecond motor actions at the multihour timescale. In the Gordus lab, in collaboration with my co-mentor, Sridevi Sarma, we have developed automated leg and web tracking algorithms that allow us to reconstruct the precise sequencing of motor actions, as well as identify the touch events between the spider and the silk that triggered a given action. These analyses have shown that the geometric stages of web- making emerge due to variations in the transition patterns among smaller motor actions (e.g., leg sweeps, locomotion), and that the triggers for these motor actions are not fixed across stages but are context-dependent. These insights suggest considerable cognitive ability in the spider, and lay the groundwork for probing the neural circuit mechanisms that give rise to these behavioral rules.

Double-strand DNA breaks are among the most toxic lesions that happen in our genomes. These lesions can cause irreversible genome modifications that have been linked with diseases such as cancer and neurodegenerative disorders as well as aging. To prevent this, our cells have developed sophisticated mechanisms to repair double-strand breaks. In Taekjip Ha’s lab, we developed a method to controllably induce double-strand breaks in human cells in high throughput. This method, which uses multitarget CRISPR (clustered regularly interspaced short palindromic repeats), enabled me to trace DNA repair processes with high temporal resolution over multiple genomic locations. In particular, I have shown how chromatin actively participates in the repair process at different length scales. In the immediate vicinity of the lesion, nucleosomes are quickly and transiently evicted, whereas at longer (megabase pair) scales, the genome undergoes extensive 3D reorganization and folds around the lesion. These findings could potentially guide the discovery of new drugs that target DNA repair pathways in diverse diseases.

Duchenne muscular dystrophy (DMD) is a lethal, X-linked, childhood-onset degenerative disease caused by mutations in the dystrophin protein, which is responsible for maintaining muscle integrity and function. The prolonged occurrence of degeneration events in dystrophic muscle leads to chronic inflammation, insufficient repair and the progressive loss of muscle mass that is replaced by fibrosis and fat, leading to significant mobility impairments and loss of ambulation. There is currently no cure for DMD, but there are drug and gene therapy efforts to convert the disease to a milder but more inflammatory form of dystrophy. One novel approach we have been studying is to modify immune cells, particularly macrophages, to prevent muscle loss and promote regeneration. Finding effective and targeted immunomodulatory treatments that can re-program inflammation and bias it toward regeneration without systemically suppressing the immune system is a major unmet clinical need. Our research aims to understand the mechanisms controlling regenerative inflammation in acute injury and dystrophy, specifically focusing on the role of recently discovered macrophage subtypes and their secreted growth factors. One of the highlights of our work is the discovery that these macrophage subsets guide the formation of structurally distinct damage-clearing and regenerative inflammation tissue zones. Our compelling high-dimensional data — single-cell and spatial transcriptomics — and advanced imaging techniques indicate that these layered regenerative tissue zones are sensitive to intermittent glucocorticoid immunosuppression (current DMD standard of care), and that they can be easily detected using a validated antibody panel and thus warrant the reevaluation of other current DMD-focused treatments on muscle regeneration. The work performed in Laszlo Nagy’s lab at the Institute for Fundamental Biomedical Research at the Johns Hopkins All Children’s Hospital highlights the importance of macrophage-organized regenerative inflammation tissue zones, and their targeting can be potentially exploited to improve outcomes in DMD.

I conducted my research in Stephen Gould’s lab, where we focus on advancing science at the intersection of organelle biogenesis and human diseases, specifically in the areas of biogenesis and engineering of small extracellular vesicles, including exosomes, viruses and virus-like particles. My research spans a broad spectrum. I have made original scientific discoveries in the areas of exosome biogenesis and mammalian cell transgenesis, advancing our understanding of a fundamental cell biological process and using these advances as a road map for the design and manufacture of exosome-based vaccines and therapeutics. Moreover, I have made significant contributions to the fight against SARS- CoV-2 by elucidating the molecular mechanisms underlying the evolution of its spike mutations. These studies revealed that the furin cleavage site insertion disrupted spike sorting and functions, while the D614G mutation acts as an intragenic suppressor of these deleterious effects. Investigation of spike trafficking also provided me with information to synergize my projects and create exosome-displayed SARS-CoV-2 Spike and influenza hemagglutinin vaccines that allow for rapid production at high efficiency and induce strong immune responses at low antigen dose.

We discovered that certain cellular factors responsible for altering the way in which the genome is packaged can find their targeted packaging elements by sliding around of DNA like a train on a track, helping these genomic factors locate their targets, which, following the train analogy, are like the destination cities. This is important because without the use of sliding on DNA, the main mechanism by which these cellular factors would find their targets is via random collision in space, which would greatly reduce their targeting efficiency. As a collaboration between the labs of my thesis adviser, Taekjip Ha, and my co-mentor, Carl Wu, we studied the dynamics of several chromatin remodelers when bound to DNA using single particle tracking on optically stretched DNA. Our discoveries are important to the field of chromatin remodeler biology because they expanded the model for how specificity and directionality of nucleosome remodeling events are encoded in eukaryotic genomes. Remodelers active on promoter proximal nucleosomes have been shown to be recruited to nucleosome depleted promoter DNA via nonspecific affinity to free DNA. We show that once bound to this DNA, remodelers perform a 1D search on DNA, and upon encountering nucleosome substrates can exhibit highly processive activity with directionality and specificity that is biased by having encountered the nucleosome via a 1D search process.

The use of gene therapy has the potential to treat a variety of diseases, including those affecting the retina. However, it’s essential to have precise control over the expression of the therapeutic gene to avoid any harmful side effects. One possible approach is to use minimal promoters and enhancers to regulate gene expression through cell-specific transcription factors. However, this strategy has drawbacks, such as unpredictable and leaky expression patterns across cell types and possible suppression during disease. We developed a novel approach called splicing-linked expression design (SLED) to address these limitations in gene therapy. SLED involves linking cell-specific splicing events with robust constitutive promoters to control gene expression. We successfully demonstrated the potential of SLED for gene therapy by creating a SLED vector that can effectively rescue photoreceptors in an animal model with retinal degeneration. We believe this approach could pave the way for more effective and safer gene therapy strategies to treat a variety of diseases.

Cerebral small vessel disease is a major cause of stroke and a significant contributor to cognitive decline and dementia. My research with the SCAN (Stroke and Cognitive Impairment Analysis Using Neuroepidemiology) lab involved a data-driven analysis of the plasma proteome to facilitate a deeper understanding of peripheral biological changes that promote small vessel disease and to identify plasma biomarkers that can noninvasively identify individuals at risk for vascular cognitive impairment and dementia. From a panel of nearly 5,000 proteins, we identified 13 that were associated with neuroimaging characteristics of small vessel disease in a large, community-based cohort. Nine of these proteins replicated in one or more external cohort and two proteins remained associated with late-life small vessel disease when measured during midlife. These findings provide the field with select plasma biomarkers and possible mechanistic mediators that can inform the development of novel therapeutic approaches.

I work in the HEPIUS (Holistic Electrical, Ultrasonic and Physiological Interventions Unburdening Those with Spinal Cord Injury) Innovation Lab with Amir Manbachi and Nicholas Theodore, where I created a technology to perform automatic detection of surgical items that may be retained in the brain during neurosurgery. For example, with hundreds of cotton balls used every day, one may accidentally be left behind and lead to potentially life-threatening immunologic responses in the patient. Cotton balls must be manually counted as they are placed in and removed from the brain, but as they soak up blood to clear the surgeon’s field of view, they become visually indistinguishable from the surrounding brain tissue. We discovered that ultrasound can discern these two materials when our own eyes cannot. I developed a deep learning algorithm with 99% accuracy, sensitivity and specificity that identifies cotton balls and other surgical items in ultrasound images of the brain. This technology is paired with a smartphone application that can be used intraoperatively without saving patient health information.

My research projects focus on the quantitative characterization of the tumor microenvironment (TME) to better understand the mechanism of cancer progression and anti- tumor immunity. Specifically, I am fascinated by the crosstalk of different cell populations within the TME in the spatial domain. Using multiplexed tissue imaging and image processing techniques, I was able to distinguish single-cell identities and their spatial locations from pathological samples of various cancer types. Using a combination of spatial statistics and machine learning, I was able to distill key spatial features that can be used to predict cancer patients’ likelihood of response to therapy and survival term. For example, my approaches revealed that the proximity of a rare subtype of myeloid — CD163- Arginase+ macrophage — to helper T cell favors improved anti-tumor immunity in hepatocellular carcinoma. Remarkably, the immunosuppressive role of myeloid cells was also observed in pancreatic cancer and non-small cell lung cancer. These findings suggest novel therapeutic candidates in immune-oncology research may benefit the design of future clinical trials and facilitate precision medicine. I conducted my research in Aleksander Popel’s lab. Our lab is devoted to applying systems biology approaches to solve problems related to cancer and ocular and cardiovascular diseases.

Neuroscientists rely on small animal  models to study the brain in exquisite detail. However, today’s brain imaging devices are massive, require anesthesia and often limit imaging to less than an hour. While miniature devices that allow brain imaging in behaving animals exist, they can only image neuronal or vascular function, not both, and only permit imaging for a few hours. These limitations significantly diminish our capacity to study brain function and restrict our understanding of the neurovascular unit and its role in brain diseases. To address this critical gap, with the mentorship of Drs. Arvind Pathak and   Nitish Thakor, I developed a new class of miniature and implantable brain imaging devices dubbed “multicontrast miniscopes,” which for the first time enables studying both neuronal and vascular activity in freely behaving animals continuously over a 24-hour period. Multicontrast miniscopes would be tools of choice for the modern neuroscientist and enable an entirely new class of neuroimaging experiments, making possible breathtaking neuroscientific discoveries.

It is remarkable that both increased and decreased dosage of the peripheral myelin protein 22 (PMP22) gene causes peripheral demyelinating neuropathy. PMP22 duplication causes Charcot-Marie-Tooth disease type 1A (CMT1A), and PMP22 deletion causes hereditary neuropathy with liability to pressure palsies (HNPP). Charcot-Marie-Tooth disease (CMT) is the leading cause of inherited peripheral neuropathy, with a prevalence of 1:2,500, and CMT1A and HNPP account for the majority of CMT cases (~62%). CMT1A and HNPP symptoms vary regarding age of onset and severity, but both diseases cause peripheral nerve deficits, which most commonly include muscle weakness, reduced sensation and neuropathic pain in distal limbs. Although CMT1A and HNPP dramatically impact patient quality of life and burden the health care system, only supportive treatments are currently available to patients. My research in the Höke lab has focused on advancing our understanding of CMT1A and HNPP pathophysiology and identifying pathomechanisms causing these diseases in order to facilitate therapy development. My results suggest that primary myelin dysfunction drives CMT1A pathogenesis because muscle atrophy occurs prior to evidence of secondary axon degeneration. Additionally, I identified Schmidt-Lanterman incisure density and organization defects in CMT1A myelin that appear to begin during development and are likely detrimental to peripheral nerve function. These findings provide important insight into CMT1A pathogenesis and reveal novel targets for designing candidate therapeutics.

We discovered the function of toxic a-synuclein (a-syn) protein aggregates in the pathogenesis of Parkinson’s disease (PD). Pathologic a-syn aggregation is a central event in Parkinson’s disease progression. Therefore, understanding the function of pathologic a-syn aggregates makes a significant conceptual advancement toward finding drug and biomarkers for this disease. We report that pathologic a-syn binds tuberous sclerosis complex 2 (TSC2) protein and disassembles TSC1-TSC2 complex that leads to persistent mTORC1 activation and mRNA translation. Inhibition of mTOR and translation by pharmacological and genetic approaches rescue pathology and neurodegeneration in PD models. I performed this research in the lab of Ted and Valina Dawson at the Institute for Cell Engineering in the Department of Neurology.

For over 38 million people worldwide living with HIV-1, antiretroviral therapy (ART) must be continued indefinitely to prevent progression to AIDS. Although ART can effectively suppress viral replication and prevent or reverse immunodeficiency in most individuals, it is not curative due to the persistence of a rare population of infected cells harboring integrated and transcriptionally inactive HIV-1 genomes referred to as the latent reservoir. A promising therapeutic approach to achieving a cure for HIV-1 is known as “shock and kill.” This approach involves selectively inducing HIV-1 gene expression (“shock”) followed by immune-mediated elimination of infected reservoir cells (“kill”). Despite therapeutic advances in reversing HIV-1 latency, no significant reduction in the latent reservoir has ever been clinically observed. Prior studies suggest that this apparent lack of reservoir elimination following latency reversal may stem from inefficient killing of reservoir cells by cytolytic immune effector cells. Therefore, to ensure effective clearance of HIV-1-infected cells in the context of “shock and kill,” novel immunotherapies must be developed to enhance HIV-1-specific cell-mediated cytolytic activity. My research in Robert and Janet Siliciano’s lab focuses on the development and functional characterization of novel bispecific antibodies, a type of immunotherapy used to enhance cell-mediated killing of specific target cell populations. These bispecific antibodies were designed to promote targeted engagement and lysis of HIV-1-infected cells by cytolytic natural killer (NK) cells. My colleagues and I have demonstrated the ability of the bispecific antibodies to mediate robust killing of HIV-1 reservoir cells using both primary cells from persons living with HIV-1 and animal models of HIV-1 infection. Thus, these bispecific antibodies represent promising preclinical candidates for therapeutic HIV-1 reservoir size reduction in combination with other agents for latency reversal.

In Justin Bailey’s lab, we study the broadly neutralizing monoclonal antibodies’ responses against hepatitis C virus (HCV) in individuals who naturally and repeatedly control infection to understand the basic biology behind successful immunity. We discovered potential vaccine antigens for the development of a successful prophylactic HCV vaccine, which is urgently needed to help achieve the World Health Organization’s goal of eliminating HCV as a public health problem by 2030.

I performed my thesis research in Mark Anderson’s lab, which studies a heart enzyme called CaMKII. Hyperactivity of this enzyme is implicated in many forms of cardiovascular disease, and thus, CaMKII blockade has become an exciting yet unexplored therapeutic strategy. CaMKII inhibitor discovery has been hamstrung by a lack of biosensors suitable for high throughput drug discovery. To solve this, we created a new sensor that reports CaMKII activity with unparalleled sensitivity and kinetics. This new tool enabled us to discover that CaMKII inhibitors already exist among drugs that are safe for human use. We found that ruxolitinib, an FDA-approved drug, is a potent CaMKII inhibitor capable of preventing models of acquired and congenital arrhythmias with minimal toxicity. Our results suggest that ruxolitinib is an ideal candidate for cardiac repurposing, and that it provides a new tool for precise measurement of CaMKII activity in living cells.

A significant challenge in neuroscience is identifying the cellular and molecular processes underlying learning and memory formation. Decades of remarkable research have found that synaptic plasticity, especially long-term potentiation (LTP), is a highly compelling cellular model of learning and memory, which requires ionotropic glutamate receptor (AMPA receptor [AMPAR]) insertion into synapses. Many AMPAR interactors have been discovered to involve the regulation of AMPAR trafficking, but less is known about their necessity in contributing to learning and memory. In Richard Huganir’s lab, my research investigates whether AMPAR interactors (GRIP1, NSF and PKMζ) are required for synaptic plasticity and memory formation, by using powerful genetic manipulations combined with electrophysiological and behavioral approaches. We have found that GRIP1 is necessary for hippocampal LTP and memory by facilitating AMPAR trafficking, and this provides insights into designing a potential therapeutic target for autism spectrum disorder. In two other studies, we proved the well-known molecules NSF and PKMζ are not required for synaptic plasticity and hippocampus-related memory. Our discoveries expand and revise the current molecular model of synaptic plasticity and help to develop comprehensive understandings of cell signaling events that contribute to learning and memory.

Just like humans, bacteria utilize immune systems to detect, evade and eliminate infectious elements such as viruses. In Dr. Joshua Modell’s lab, we study many bacterial immune mechanisms, but my research focuses on the prokaryotic adaptive immune system CRISPR-Cas. The quandary that drove my thesis work is how bacteria can balance the benefits of immunity with the inevitable costs of carrying an immune system. We discovered that some bacteria repurpose the immune effector Cas9 to auto-regulate the production of the CRISPR-Cas system components. Furthermore, we found that this auto-regulatory mechanism, although it reduced immunity overall, was ultimately beneficial to the cells in minimizing autoimmune costs. Our work reveals an important mechanism through which bacteria can stably maintain immune systems while providing insights into novel functions of Cas9, as well as insight into regulators that could shape next-generation Cas9 therapeutic applications.

In Dr. G. William Wong’s lab, I uncovered a biological process that can effectively prevent high-fat diet-induced weight gain and progression toward type 2 diabetes. While initially studying the effects trisomy of the 21st chromosome (Down syndrome) has on systemic metabolism and the development of metabolic disease, I serendipitously discovered the presence of an extremely strong futile cycle within the skeletal muscle of one of our mouse models. Quite amazingly, the presence of this particular futile cycle directly promotes continuous energy (ATP) depletion. This, in turn, drives the energy production pathways of the body, which results in massive calorie utilization and energy expenditure. The resultant organism is almost completely resistant to gaining weight and does not develop many of the hallmark signs of type 2 diabetes. This work provides very helpful insight and proof-of-concept to this anti- obesity pathway, which could be harnessed as a treatment for obesity and diabetes.

Wilson disease (WD) is a metabolic disorder of copper (Cu) homeostasis that can present significant diagnostic and treatment challenges. Despite the well-established cause, the mechanisms behind WD pathologies remain only coarsely defined, hindering enhanced therapies’ development. At the Lutsenko lab in the Department of Physiology, we have identified specific molecular links connecting Cu overload to changes in the activity of liver X receptor (LXR) in the mouse model of WD. In Atp7b−/− mice with established liver disease and human WD, Cu overload activates the stress-sensitive transcription factor Nrf2. Nrf2 targets, especially sulfotransferase 1e1 (SULT1E1), are strongly induced and cause elevation of sulfated sterols, whereas oxysterols are decreased. This sterol misbalance results in the inhibition of the LXR and upregulation of LXR targets associated with inflammatory responses. Pharmacological inhibition of SULT1E1 partially reverses oxysterol misbalance and LXR inhibition. Contribution of  this pathway to advanced hepatic WD was demonstrated by treating mice with an LXR agonist. We further showed that after the onset of metabolic changes, inflammation, and fibrosis in the WD liver, treatment with an LXR agonist can lessen these manifestations. Thus, our findings indicate that the identified pathway is an important driver of WD pathogenesis downstream of elevated Cu. Modulation of LXR activity should be investigated as a therapeutic approach to supplement chelation in WD.

The clinical motivation for our work in Chulan Kwon’s lab is the need for cardiac cells for regenerative therapies, particularly for patients who have suffered heart attacks and have damaged heart muscle, as well as for drug screening purposes. One highly promising approach under investigation is the use of cardiac cells derived from pluripotent stem cells (PSCs), including patient- derived induced PSCs. However, PSC-derived cardiac cells have thus far been immature, resembling fetal instead of adult cardiac tissue. This, in turn, has significantly limited their clinical use. My project studied developmental processes underlying cardiac maturation, and identified a transcription factor regulatory network that controls this maturation process. We are hopeful that by targeting this network in the future, we can develop mature cardiac tissues that can benefit patient cardiac health.

Cell migration is a fundamental process that plays a crucial role in a wide array of physiological and developmental scenarios, such as immune response, wound healing, stem cell homing and embryogenesis. On the other hand, its dysregulation often results in different detrimental outcomes, including autoimmune diseases, cognitive deficits and cancer metastasis. As an upshot of extensive research for the past three decades, many signal transduction and cytoskeletal molecules have been identified that collectively work to sense and process different external cues, generate proper polarity and help the cell to correctly navigate via coordinated protrusions and contractions. While numerous specific interactions among many such signaling and cytoskeletal molecules have been deduced through biochemical and genetic analyses, how the activities of so many different components are spatially and temporally coordinated at the subcellular scale has remained unclear. In other words, little was known about the global scale organization mechanisms that determine when and where the next protrusions will form or how polarity will be organized in a migrating cell. My research, carried out in the labs of Peter N. Devreotes and Pablo A. Iglesias, answered these fundamental questions on cell migration and signal transduction. We demonstrated that the dynamic regulation of inner membrane surface potential is sufficient and necessary to regulate the cell polarity and migration mode. We developed novel monitoring tools and optogenetic actuators that can work in conjunction with standard live-   cell imaging setup and genetic/pharmacological perturbations. Using these systems, we established that surface charge on the inner leaflet of the plasma membrane, a biophysical property — not some coincidental congruence of stepwise specific biomolecular interactions — spatially and temporally orchestrate signal transduction activities in the cell to control protrusion formation. Our experiments in Dictyostelium amoeba and different mammalian cells demonstrated that surface charge is dynamically altered during signaling network activation and, in turn, its generic perturbation can induce or inhibit signaling activities that mediate cell migration. It is well known that during propagation of nerve impulse, transmembrane potential can regulate the opening of the specific ion channels, which in turn collectively define the transmembrane potential. Our results indicated that transiently lowered inner membrane surface potential, which we termed “action surface potential,” can analogously propagate and interact with signaling network activation.

The mechano-electrical transduction  (MET) by the sensory hair cells of the inner ear is crucial for sound perception. Over the last several decades, studies on hair cell MET have demonstrated that the channel machinery is assembled from an astonishing number of diverse molecules, yet the molecular and cellular functions of those proteins in MET remain uncharacterized. I’ve been working in the laboratory of Uli Mueller in the Department of Neuroscience, where my research has focused on uncovering the mechanisms by which various MET channel subunits assemble and cooperate to sense mechanical stimuli in cochlear hair cells. We believe the studies are fundamentally important for understanding the molecular mechanism of sound sensation in physiological and pathological conditions.

Microtubules, a dynamic network of protein filaments, are involved in many essential cellular functions, and the dysregulation of microtubule regulation is often associated with a wide range of human diseases, such as cancer and neurodegeneration. Because of their unique hollow structure, microtubules have luminal space, and there is accumulating evidence that microtubule properties are modulated by protein interactions on their luminal surface. This raises a primitive question: How can proteins enter the microtubule lumen, which is surrounded by densely packed tubulin dimers? Under the mentorship of Dr. Takanari Inoue, I developed “luminal molecular trapping,” which for the first time allowed for real-time visualization of molecular accessibility to microtubule lumens. By utilizing this technique, I discovered that soluble proteins efficiently enter the lumen through their peripheral ends and side lattice openings. The present luminal trapping strategy laid the foundation for probing luminal microtubule biology and has potential in extending the study to uncovering regulations of effector proteins and tubulin post-translational modifications.